Docetaxel is the first-line treatment for castration-resistant prostate cancer (CRPC). The limited survival benefit associated with the quick emergence of resistance and systemic toxicity diminishes its efficacy in high-dose monotherapy. YK-4-279 is a small molecule inhibitor of ETV1 that plays an important role in the progression of prostate cancer.
Int J Med Sci 2017, Vol 14 Ivyspring International Publisher 356 International Journal of Medical Sciences 2017; 14(4): 356-366 doi: 10.7150/ijms.18382 Research Paper The Effects and Mechanism of YK-4-279 in Combination with Docetaxel on Prostate Cancer Lin Yu1, Xiaofeng Wu1, Min Chen1, Huarong Huang1, Yan He1, Huaqian Wang1, Dongli Li2, Zhiyun Du1, Kun Zhang1, 2, Susan Goodin3, Xi Zheng1, 4 Allan H Conney Laboratory for Anticancer Research, School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, China; School of Chemical and Environmental Engineering, Wuyi University, Jiangmen 529020, China; Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08903, USA; Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA Corresponding author: Xi Zheng, Allan H Conney Laboratory for Anticancer Research, School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, China; Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA Phone: 848-445-8069 Email: xizheng@pharmacy.rutgers.edu © Ivyspring International Publisher This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/) See http://ivyspring.com/terms for full terms and conditions Received: 2016.11.16; Accepted: 2017.03.14; Published: 2017.04.07 Abstract Background: Docetaxel is the first-line treatment for castration-resistant prostate cancer (CRPC) The limited survival benefit associated with the quick emergence of resistance and systemic toxicity diminishes its efficacy in high-dose monotherapy YK-4-279 is a small molecule inhibitor of ETV1 that plays an important role in the progression of prostate cancer The aim of this study was to evaluate the hypothesis that the combination of docetaxel and YK-4-279 will have a synergistic effect on inhibiting growth and accelerating apoptosis in human prostate cancer cells Methods: Cell growth assessed using CCK-8 and trypan blue exclusion assays Cell apoptosis was determined by morphological assessment in cells stained with propidium iodide Standard scratch migration and Matrigel-coated transwell invasion assays were used to assess cell migration and invasion, respectively Western blotting was used to investigate the levels of ETV1, AR, PSA, p-STAT3, survivin, Bcl-2, and p-Akt in prostate cancer cells Results: The combination of low-dose docetaxel and YK-4-279 synergistically inhibited growth and induced apoptosis in human prostate cancer cells The combination also more efficiently suppressed the migration and invasion of LNCaP and PC-3 cells The combination of low-dose docetaxel and YK-4-279 caused a stronger decrease in the levels of ETV1, AR, PSA, p-STAT3, survivin, Bcl-2, and p-Akt in LNCaP cells and of p-Akt, Bcl-2, and p-STAT3 in PC-3 cells compared with either drug alone Conclusions: These data suggest that the combination of docetaxel and YK-4-279 may be an effective approach for inhibiting the growth and metastasis of prostate cancer This could permit a decrease in the docetaxel dose necessary for patients with CRPC and thereby lower its systemic toxicity Key words: docetaxel, YK-4-279, prostate cancer, combination, synergistic action Introduction Prostate cancer is the most frequently occurring cancer and the second leading cause of cancer-related deaths among men in the United States [1] Patients diagnosed with localized stage disease are sensitive to various treatments and are often curable; however, ~40% of all cases will eventually progress to a metastatic stage [2] Since the 1940s, targeting androgen signaling using androgen deprivation therapy (ADT) has been the mainstay of clinical interventions for metastatic hormone-sensitive prostate cancer [3] However, its initial effectiveness is only transient (2–3 years) [4], http://www.medsci.org Int J Med Sci 2017, Vol 14 and most men relapse with castration-resistant prostate cancer (CRPC) and die soon thereafter [5] For patients with CRPC, the common treatment is docetaxel-based chemotherapy to prolong survival and maintain a good quality of life [6] However, treatment using high doses of docetaxel ultimately cause toxicity and resistance, which means that there are only limited options for patients progressing on or after docetaxel [7] Recently, some novel agents including abiraterone, cabazitaxel, and enzalutamide were approved for patients with CRPC following docetaxel failure or resistance, but their efficiency is limited [8] Therefore, there is an urgent need to improve docetaxel-based regimens to reduce toxicity and increase efficacy Prostate cancer that progresses to lethal CRPC has been associated with ETS gene fusions, PTEN loss, and androgen receptor (AR) amplification [9] In particularly, ETS transcription factor (mainly ETV1 or ERG) fusions occur frequently in prostate cancer [10-11], and ~50% of human prostate cancers containing ETS gene fusions [12] One study suggested that ETV1 expression promotes autonomous testosterone production to reactivate AR signaling in aggressive disease [13] It was suggested that ETV1 plays an important role in the progression of CRPC and can indirectly mediate AR signaling YK-4-279 monotherapy can inhibit the growth and metastasis of ETV1 fusion-positive prostate cancer xenografts [14-15] This suggests that YK-4-279 could be used as a small molecule inhibitor of ETV1 Docetaxel, a semi-synthetic second-generation taxane, can slow down the progression of prostate cancer, and it retains antitumor activity in CRPC patients [16] It can inhibit proliferation and induce apoptosis by binding to β-tubulin and causing cell-cycle arrest [17] The addition of docetaxel to ADT has survival benefits compared with ADT alone in patients with metastatic hormone-sensitive prostate cancer [18-19] This finding suggests that there is an interaction between AR signaling and docetaxel sensitivity Thus, we hypothesized that the combination of YK-4-279 and docetaxel will synergistically inhibit growth and accelerate apoptosis in human prostate cancer cells 357 dissolved in DMSO (Sigma, USA); the final concentration of DMSO was 0.1% in all experiments Cell viability For CCK-8 assays [21], cells were seeded at a density of × 10 cells/ml of medium in a 96-well plate (0.1 ml/well) and incubated for 24 h Then, the cells were treated with different concentrations of docetaxel (1 nM) and YK-4-279 (0.1 µM, 0.5 µM, or 1.0 µM) for 72 h After treatment, the media were replaced with fresh media and 10 µl CCK-8 (Dojindo, Kumamoto, Japan) was added to each well After a 1-h incubation, the absorbance at 450 nm was measured on a microplate reader For the trypan blue exclusion assays [22], cells were seeded at a density of × 104 cells/ml of medium in 35-mm tissue culture dishes (2 ml/dish) and incubated for 24 h The cells were then treated with different concentrations of docetaxel (0.1 nM, 0.5 nM, or nM) and YK-4-279 (0.1 µM, 0.5 µM, or 1.0 µM ) for 72 h Then, single cell suspensions were prepared and the number of viable cells was counted using a hemocytometer under a light microscope by mixing 80 µl of cell suspension and 20 µl of 0.4% trypan blue solution for Blue cells were counted as dead and the cells that did not absorb dye were counted as live Measuring apoptosis Materials and methods Apoptosis was determined by morphologically assessing cells stained with propidium iodide (PI) [22] Cells were seeded at a density of × 104 cells/ml in 35-mm tissue culture dishes (2 ml/dish) and incubated for 24 h They were then treated with docetaxel (0.5 nM) and/or YK-4-279 (0.5 µM) for 72 h After treatment, cytospin slides were prepared using a smear centrifuge and fixed with acetone/methanol (1:1) for 10 at room temperature The cells were stained with µg/ml PI in PBS for 10 Then we identified apoptotic cells using a fluorescence microscope Cells with classical morphological features including nuclear condensation, cell shrinkage, and the formation of apoptotic bodies were counted as apoptotic At least 400 cells in approximately 10 randomly selected fields were counted in each sample Cell culture and reagents Scratch migration assays Two human prostate cancer cell lines (LNCaP and PC-3) were obtained from the American Type Culture Collection (Rockville, MD, USA) YK-4-279 was purchased from MCE (MedChem Express, HY-1450, USA) and docetaxel was from Aladdin (D107319) The cells were cultured as described previously [20] Docetaxel and YK-4-279 were For scratch migration assays [23], cells were seeded at a density of × 105 cells/ml in 35-mm tissue culture dishes (2 ml/dish) and incubated for 24 h The cell surface was then scratched using a sterile 200-µl pipette tip (Axygen, Union City, CA, USA) after washing with PBS Then, the scratched cells were rinsed gently with PBS three times and complete medium was added (0.2% FBS) The cells were then http://www.medsci.org Int J Med Sci 2017, Vol 14 treated with different concentrations of docetaxel and/or YK-4-279 for an additional 24 h Images were captured using an inverted microscope The distance that cells migrated compared with baseline measurements was measured using Image J software Invasion assays For Matrigel-coated transwell invasion assays [24], 600 µl complete medium (20% FBS) was added to the lower chamber The cells were pre-treated with YK-4-279 or docetaxel for 48h Then, the cells were trypsinized, resuspended in complete medium (1% FBS), and seeded at a density of × 105 cells/ml in the top chamber (200 µl/chamber; Corning) containing a Matrigel-coated membrane The cells were then treated with different concentrations of docetaxel and/or YK-4-279 for an additional 24 h Next, the medium and the cells remaining in the top chambers were removed After fixing with methanol and staining with 0.1% crystal violet, the number of cells that had invaded to the lower membrane was counted and images were captured under an inverted microscope (Olympus) Western blotting After treatment, the protein lysates were prepared as described previously [25] Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) After blocking nonspecific binding sites with blocking buffer, the membranes were incubated overnight at 4˚C with the following primary antibodies: #4060 for phospho-Akt, #9145 for phospho-Stat3, #5365 for PSA/KLK3, #2808 for survivin, #2870 for Bcl-2, #4370 for phospho-p44/42 MAPK (ERK-1/2), #12153 for IL-6, #3202 for AR (all from Cell Signaling Technology, Beverly, MA), and ab81086 for ETV1 (Abcam, Cambridge, MA, USA) β-actin (Cell Signaling Technology, Beverly, MA) was used as a loading control Following removal of the primary antibody, the membranes were washed three times with TBST (TBS containing 0.05% Tween 20) at room temperature and then incubated with fluorochrome-conjugated secondary antibody for h The membranes were then washed with TBST three times and signals were detected using ECL in the dark room Statistical analysis The potential synergistic effects of docetaxel and YK-4-279 were assessed using the isobole method with the equation Ac/Ae + Bc/Be = combination index (CI) [26] Ac and Bc represent the concentration of drug A and drug B used in the combination, respectively, and Ae and Be represent the 358 concentration of drug A and B that produced the same magnitude of effect when administered alone If CI is 1 or =1, then the drugs act in an antagonistic or additive manner, respectively Comparisons of cell viability, apoptosis, migration, and invasion were analyzed using ANOVA with Tukey-Kramer multiple comparison tests Results Effects of docetaxel and YK-4-279 alone or in combination on prostate cancer cell growth and apoptosis The effects of docetaxel and YK-4-279 alone or in combination on the growth of human prostate cancer cells were determined using the CCK-8 and trypan blue exclusion assays Human LNCaP (androgen-dependent), PC-3 (androgen-independent) prostate cancer cells were treated with different concentrations of docetaxel and YK-4-279 for 72 h As shown in Fig 1A and B, treatment with YK-4-279 (0.1 µM, 0.5 µM, and 1.0 µM) alone and in combination with docetaxel (1 nM) inhibited the growth of both LNCaP and PC-3 cells in a dose-dependent manner Treatment with the combination of docetaxel and YK-4-279 had a stronger inhibitory effect on cell growth than either drug alone When the concentration ratio of YK-4-279 and docetaxel was 1:1000, co-treatment with YK-4-279 and docetaxel exhibited a stronger decrease in cell viability compared with the other two combination groups As shown in Fig 1C and D, docetaxel and YK-4-279 single and combined treatments affected the viability of LNCaP and PC-3 cells in a dose-dependent manner The half maximal inhibitory concentrations (IC50) of YK-4-279 were 1.48 µM and 2.03 µM in LNCaP and PC-3 cells, respectively, and the IC50 of docetaxel alone was 1.16 nM and 2.07 nM in LNCaP and PC-3 cells, respectively Specifically, the IC50 of docetaxel was decreased to 0.41 nM and 0.66 nM in LNCaP and PC-3 cells respectively, whereas those of YK-4-279 decreased to 0.41 µM and 0.66 µM The combination indexes (CIs) for the IC50 were 0.64 and 0.64 in LNCaP and PC-3 cells, respectively These results suggest that the combination of YK-4-279 and docetaxel synergistically inhibited the growth of both LNCaP and PC-3 cells Next, the effects of docetaxel and YK-4-279 alone or in combination on prostate cancer cell apoptosis were determined by morphologically assessing cells stained with PI Apoptotic cells were identified according to classic morphologic features such as nuclear condensation, cell shrinkage, and the formation of apoptotic bodies [23] Morphologically http://www.medsci.org Int J Med Sci 2017, Vol 14 distinct apoptotic cells from representative samples are shown in Fig As shown in Table 1, treating cells with docetaxel (0.5 nM) and YK-4-279 (0.5 µM) alone resulted in a small number of apoptotic cells In contrast, the combination of docetaxel and YK-4-279 caused a strong increase in the number of apoptotic cells, suggesting that the combination of docetaxel and YK-4-279 had a more potent effect on stimulating apoptosis than either agent alone 359 Table Effect of docetaxel and YK-4-279 alone or in combination on LNCaP and PC-3 cell apoptosis Treatment Apoptotic cells (%) LNCaP PC-3 Control 2.32±0.31 3.38±0.89 Docetaxel 4.95±0.57 4.38±0.86 YK-4-279 5.52±0.84 4.72±0.81 Combination 17.03±3.12*** 17.04±0.64*** Dead cells (%) LNCaP 3.67±1.15 5.33±0.58 6.67±0.58 18.67±0.15*** PC-3 4.01±0.55 4.88±0.78 4.94±0.38 19.96±2.11*** LNCaP or PC-3 cells was cultured at a density of × 10 cells/ml for 24 h The cells were then treated with docetaxel (0.5nM) and YK-4-279(0.5µM) alone or in combination for 72h Apoptosis was determined by morphological assessment Each value represents mean±S.E from three separate experiments Significant numbers of apoptotic cells between a combination group and a single-agent-treated group were analyzed by ANOVA with Tukey-Kramer multiple comparison test (*p